Summary

The glomerulus of the mammalian kidney is an intricate structure that
contains an unusual filtration barrier that retains higher molecular weight
proteins and blood cells in the circulation. Recent studies have changed our
conception of the glomerulus from a relatively static structure to a dynamic
one, whose integrity depends on signaling between the three major cell
lineages: podocytes, endothelial and mesangial cells. Research into the
signaling pathways that control glomerular development and then maintain
glomerular integrity and function has recently identified several genes, such
as the nephrin and Wilms' tumor 1 genes, that are mutated in human kidney
disease.

Introduction

The glomerulus of the mammalian kidney is a highly developed vascular bed
that acts as a filter, allowing a filtrate of small molecules, such as water,
sugars, electrolytes and small proteins, to pass through a barrier that
retains high molecular weight proteins and cells in the circulation. The
proper development and preservation of this structure throughout life is
essential to the prevention of serious disease. The past ten years have
witnessed numerous advances in our understanding of glomerular development and
function. Podocytes, the visceral epithelial cell of the glomerulus, are now
recognized as being a key cell type, the injury of which can initiate
glomerular scarring. Several genetic kidney disorders are caused by mutations
in genes that encode proteins that appear to have highly specialized functions
in podocytes, especially in the maintenance of the protein barrier, which
prevents massive protein loss from the circulation (a condition known as
nephrotic syndrome). The glomerular basement membrane and its receptors have
also served as one of the key models for the study of how a basal lamina
develops and interacts with adjacent epithelial cells. Moreover, glomerular
research has added to our understanding of how signals between adjacent cell
types are required for the proper development and maintenance of the
structural integrity of an organ throughout life.

The de novo regeneration of an entire nephron or a whole glomerulus has
never been documented in mammals. Indeed, aside from repairing proximal
tubules damaged in acute situations, the kidney has a very limited ability to
repair itself compared with many other organs. Because the glomerulus only
develops in the context of the induction of an entire new nephron during
kidney development, it is unlikely that we will learn how to regenerate
glomeruli, except in the context of discovering how to regenerate entirely new
nephrons. While this remains a long term goal of kidney development research,
perhaps a more accessible therapeutic target will be the podocyte, where an
ability to restore foot process architecture has the potential to reduce
dramatically the morbidity and mortality that results from chronic kidney
disease.

In this review, we focus on recent advances in glomerular development and
biology, and relate them to the disease processes, possible avenues of
treatment and the prevention of end-stage kidney disease that these advances
have opened up.

Kidney development and glomerular formation

Nephron induction

Each human kidney contains approximately one million nephrons. The
glomerulus is the most proximal component of the nephron (see
Fig. 1). The segmentation of
the nephron (Fig. 1C) presents
a fascinating, but poorly understood, process. It is becoming clear that
signaling via the Notch pathway, particularly through NOTCH2 and its ligands,
is involved in this segmentation process
(Cheng and Kopan, 2005;
Cheng et al., 2003;
Leimeister et al., 2003;
McCright et al., 2002).
Blocking Notch signaling in mouse embryonic kidney organ culture interferes
with the development of the proximal components of the nephron, including the
glomeruli and proximal tubules, although it does not block the differentiation
of podocytes, a major cell type of the glomerulus, if they (or the glomerulus)
have been specified prior to initiating the Notch blockade
(Cheng and Kopan, 2005;
Cheng et al., 2003). Moreover,
the conditional deletion of Notch2 from nephron progenitor cells in
the developing mouse kidney results in a `distal tubule only' phenotype, as
the proximal tubules and glomeruli are absent in these mutants
(Cheng et al., 2007). Thus, it
appears that Notch signaling may be involved in establishing the major
proximodistal axis of the nephron, but is less important in the specification
of the glomerulus itself.

Glomerular formation: podocyte differentiation is the first
determinant

The precursor structure of the glomerulus can be first appreciated in the
`S-shaped body', so-called because it is shaped like an `S' when observed in
histological sections. There are three major components to the early
glomerulus (Fig. 2): the layer
of primitive podocytes that begins as a columnar epithelium; the thin layer of
Bowman's capsule, which appears to be nearly flat, similar to a squamous
epithelium; and the capillary loop that first enters the glomerular cleft
(Fig. 2A,B). How the podocytes
extend themselves around the capillary loops remains unknown. Early in this
process, both the podocytes and the capillary endothelial cells form their own
basal lamina. As the glomerulus matures, these two basal lamina fuse to form a
thick basement membrane, known as the glomerular basement membrane (GBM)
(Fig. 2E). Concomitant with
this fusion, which brings the podocytes and endothelial cells into close
apposition with each other, the podocytes undergo a remarkable transformation,
during which they acquire some mesenchymal-like characteristics, although they
remain an atypical epithelial cell. The podocytes begin to lose their lateral
cell attachments, except at a point immediately adjacent to the basal membrane
(Fig. 3). They also extend
themselves nearly completely around the capillary loops. Finally, mature
podocyte cell bodies that have become isolated from each other, extend several
large projections, each of which divides into intermediate branches, which
then divide into many smaller `foot processes' that interdigitate with the
foot processes of adjacent podocytes (Fig.
2D).

A schematic of kidney development. (A) Cross section of an
E11.5 mouse embryonic kidney at induction. Mesenchyme (blue) condenses around
the two branches of the ureteric bud (UB, red), and receives an inductive
signal from it, which includes Wnt9b
(Carroll et al., 2005).
(B) Some of the mesenchymal condensate forms a pre-tubular aggregate
(PTA) adjacent to the underside of each branch of the UB. This aggregate
undergoes a mesenchymal to epithelial transformation, under the influence of
Wnt4 (Stark et al., 1994), to
form a simple tubule called a renal vesicle (RV). (C) The RV then
undergoes segmentation to form the nephron, which consists of the glomerulus
(G) at the proximal end, and the tubular component [the proximal (P) tubule,
the ascending and descending loops of Henle (not shown) and the distal (D)
tubule]. The distal tubule connects to the UB, which itself transforms into
collecting ducts that conduct urine out of the kidney. (D) A mature
nephron (not to scale), showing capillary loops (red) inside the glomerulus,
and the glomerular basement membrane (GBM, green) between podocytes (blue) and
the capillaries (mesangial cells are not shown). The distal segment of the
nephron (light blue) connects to a collecting duct (red) that is derived from
the UB. (E) To form the mature kidney, the process shown in A-D is
reiterated by continued branching of the UB and its derivatives (red). Each of
the tips of the UB derivatives continue to induce new nephrons (blue) from a
population of progenitor cells present at the periphery of the developing
kidney, known as the nephrogenic zone (NZ). Nephrons are located in the cortex
(unshaded; with some segments dipping into the medulla), whereas collecting
ducts (red) derived from the UB extend from the cortex to the medulla (green)
and the medullary papilla (pink), where they drain into the ureter.
Reproduced, with permission, from Kreidberg
(Kreidberg, 2006).

Foot process assembly

The inter-digitation of podocyte foot processes around the capillaries is a
unique aspect of glomerular development that is fundamental to the maintenance
of renal function and the prevention of glomerular disease. The basal aspect
of the foot processes adhere to the GBM, and, where they retain their
cell-cell contacts, they form a cell-cell junction, called the slit diaphragm,
which is discussed in detail below. The appearance of these cellular
extensions and interdigitated foot processes is indicative of a process in
which cell bodies that have become isolated from each other, extend processes
towards each other, that then become interdigitated as they envelope the
capillaries. Alternatively, they might remain attached at their lateral faces,
as described above, and remodel their cell-cell junctions into the projections
that acquire the appearance of foot processes
(Fig. 3). This model suggests
that the major and intermediate projections between the cell bodies and the
foot processes arise passively as a consequence of the separation of
podocytes, which only remain attached to each other where foot processes
interdigitate. There is, at present, little understanding of how this might
occur, although some insight may be gained from a study of keratinocytes, in
which an intermediate step in the formation of a cell-cell junction was
reported to involve the interdigitation of filopodial processes
(Vasioukhin et al., 2000).
Unfortunately, the small size of foot processes requires that they are studied
by electron microscopy, and the lack of a suitable in vitro model that
displays interdigitating foot processes makes the real-time analysis of foot
process assembly very difficult, if not impossible, with currently available
imaging technology.

Glomerular vascular development: mesangial and endothelial cells

Glomerular capillary development begins when a single capillary loop grows
into the glomerular cleft, which is situated between the primitive podocytes
and the proximal tubule of the S-shaped body (Figs
1,
2). As glomerular maturation
proceeds, the capillary loop becomes divided into six to eight loops
(Potter, 1965). The
endothelial cells acquire a fenestrated morphology, such that there are
slit-like openings on both sides of the GBM: on the podocyte side there are
slits between adjacent foot processes, and on the endothelial side, the slits
are actually through the endothelial cells themselves (Figs
2,
4).

Mesangial cells are found adjacent to endothelial cells on the opposite
side of the GBM from podocytes (Figs
2,
4). Some studies indicate they
originate from the mesenchymal precursors that contribute to the other cells
of the nephron, whereas others suggest an extra-renal origin, perhaps from a
component of the hematopoietic lineages
(Abe et al., 2005;
Masuya et al., 2003;
Takeda et al., 2006). They
are mostly found in the stalk of the glomerular tuft, where they possibly help
to maintain the structure of the capillary loops. Mesangial cells share
similarities with pericytes and smooth muscle cells, and thus, may help the
glomerular vasculature respond to various physical stimuli
(Schlondorff, 1987;
Yamanaka, 1988). Moreover, in
some forms of glomerular disease referred to as `diffuse mesangial sclerosis'
(DMS), there is an accumulation of extracellular matrix (ECM) on the vascular
side of the GBM, of which mesangial cells are presumed to be the origin, which
eventually forms scar tissue that can replace capillary loops and dramatically
interfere with renal function. Interestingly, mutation of the Wilms' tumor 1
(WT1) gene, which is expressed in podocytes, is one of the most
well-characterized situations that leads to DMS
(Denys et al., 1967;
Drash et al., 1970;
Habib et al., 1985). This
finding, and others discussed in the following sections, has led to the
paradigm that interactions between podocytes and mesangial and endothelial
cells are essential for maintaining normal glomerular structure and function
throughout life.

When mature, the glomerular capillary `tuft' (see
Fig. 4) consists of several
capillary loops with mesangial cells at their base, some of which extend into
each branch of the capillary structure
(Potter, 1965). The entire
tuft is enveloped by the GBM, and podocytes extend their foot processes around
these capillary loops.

The glomerular basement membrane

The GBM is a specialized basal lamina and is an important component of the
protein barrier that prevents high molecular weight proteins from leaving the
circulation while transiting the glomerular capillary bed. The major
components of the GBM are type IV collagen, laminin, and the heparan sulfate
proteoglycan agrin (Miner,
1999). The earliest epithelial cells of the nephron mainly express
laminin 1 (α1β1γ1). As soon as it is possible to define a
nascent GBM, a shift in laminin expression occurs to isoforms that contain theα
4 subunit (laminin 8: α4β1γ1). Upon further maturation
of the GBM in the S-shaped body, there is a second shift to the expression of
laminin 10 (α5β1γ1). At the capillary loop stage, laminins 9
(α4β1γ1) and 11 (α5β2γ1) are found, but in
the mature glomerulus, laminin 11 is the only laminin isoform present in the
GBM (Abrahamson and St John,
1993; Durbeej et al.,
1996; Ekblom et al.,
1991; Miner, 1998;
Miner et al., 1995;
Miner and Yurchenco, 2004;
Sorokin et al., 1997). There
is also a shift in the expression of type IV collagen
(Miner and Sanes, 1994). The
early nephron mainly expresses the α1 (IV) and α2 (IV) collagen
subunits and, upon maturation of the GBM, there is a shift to α3,α
4 and α5 (IV) subunits (Miner
and Sanes, 1994).

The slit diaphragm

The identification of nephrin as the product of the NPHS1 gene,
which is mutated in the Finnish form of Congenital Nephrotic Syndrome
(Holthofer et al., 1999;
Kestila et al., 1998;
Lenkkeri et al., 1999),
renewed attention on the slit diaphragm (SD) as a structure that is involved
in maintaining normal renal function, the damage of which may be involved in
the initiation and progression of glomerular disease, leading to dialysis and
transplantation. The assembly of the SD is an important part of glomerular
development, as it is integral to the assembly of correctly and interdigitated
podocyte foot processes. The SD, which is only visible by high power electron
microscopy, is a structure that connects adjacent foot processes. It consists
of a complex of proteins that serves as a component of the protein barrier
(Hamano et al., 2002;
Tryggvason and Wartiovaara,
2001) (Fig. 5). The
relative importance of the endothelial layer versus GBM versus the SD in
preventing proteins from exiting the circulation is a matter of long-standing
debate; most probably they all have an integral role. In the pediatric
setting, in conditions such as Congenital Nephrotic Syndrome, the SD never
develops properly (Holthofer et al.,
1999; Ruotsalainen et al.,
1999), and infants born with this condition require intensive
support early in life, including dialysis. Similarly, mice with targeted
mutations in the Nphs1 gene encoding nephrin also fail to survive
beyond the first day or two after birth
(Hamano et al., 2002;
Putaala et al., 2001;
Rantanen et al., 2002). Three
other genes that encode proteins with structural similarity to nephrin,
Neph1, Neph2 and Neph3, have been identified that appear to
interact with other slit-diaphragm proteins similarly to nephrin (Donoviel et
al., 2001; Gerke et al., 2005;
Sellin et al., 2002). A gene
trap mutation in Neph1 leads to glomerular disease in mice (Donoviel
et al., 2001). There is evidence of both homophilic interactions between
nephrin molecules and heterophilic interactions between nephrin and members of
the Neph family (Barletta et al.,
2003; Gerke et al.,
2005; Khoshnoodi et al.,
2003; Liu et al.,
2003). At least some portion of the SD can also be viewed as the
cell-cell junction that is retained between adjacent podocytes
(Reiser et al., 2000;
Ruotsalainen et al., 2000).
In support of this notion, P-cadherin and the protocadherin FAT1 have been
discovered in the SD (Inoue et al.,
2001; Reiser et al.,
2000). Although mutation of the P-cadherin gene in mice does not
result in any glomerular abnormalities
(Radice et al., 1997), a null
mutation in Fat1 results in a failure to form foot processes
(Ciani et al., 2003).
Together, these results raise the question of whether some proteins, such as
nephrin and associated proteins (discussed below), primarily serve as a
protein barrier, possibly by acting as a repulsive force to maintain a small
distance between adjacent foot processes, whereas others, such as P-cadherin
and FAT1, serve as structural components of the cell-cell junction. In support
of this is the observation that foot processes are not immediately lost in
nephrin-deficient mice (Hamano et al.,
2002; Putaala et al.,
2001; Rantanen et al.,
2002), although the SD is no longer detectable by electron
microscopy.

A theoretical model of podocyte maturation and foot process
assembly. (A) Two podocytes (blue) begin as discs of columnar
epithelial cells, which are attached along their entire lateral membranes.
(B) Podocytes lose their lateral cell attachments except at their base,
and begin to interdigitate along the basal aspect of the lateral membrane.
(C) Podocyte cell bodies become independent of each other, but remain
attached through interdigitated foot processes.

Schematic of a mature glomerulus in cross section. Fewer capillary
loops are shown than normal for clarity, and the size of cells are exaggerated
in proportion to the overall size of the glomerulus. The four major cell types
of the glomerulus are the Bowman's capsule (BC) or parietal epithelium (gray),
podocytes (P, blue) or visceral epithelium, mesangial cells (M, orange) and
endothelial cells (E, red). The mature glomerulus is encompassed by the
Bowman's capsule. The glomerulus comprises a self-contained network of
capillary loops (C, red), with mesangial cells forming a nexus at the base of
the capillary network. The glomerular basement membrane (GBM, green) divides
the glomerulus into two compartments, an inner one containing the capillaries
and the mesangial cells, and an outer one containing podocytes and the space
into which the filtrate passes. The glomerulus remains connected to the
remainder of the nephron through an opening in the Bowman's capsule that
connects the glomerulus to the proximal tubule, shown on the right. The arrows
in the capillaries indicate the flow of blood in and out of the glomerulus.
Also omitted for clarity is the branching of the single capillary loop into
the multiple loops within each glomerulus.

Molecular regulation of podocyte function

The first marker of glomerular development in vertebrates is the
restriction of Wt1 expression to a subset of cells within the renal
vesicle, as it is transforming through the comma to S-shaped tubule
(Armstrong et al., 1993;
Pelletier et al., 1991b).
Several other transcription factors are expressed in the early podocytes
within S-shaped tubules, including podocyte-expressed 1 (Pod1; also
known as capsulin; Tcf21 - Mouse Genome Informatics)
(Quaggin et al., 1998b),
forkhead box C2 (Foxc2)
(Takemoto et al., 2006),
kreisler (Mafb) (Sadl et al.,
2002), the forkhead domain transcription factor Mf2
(Foxd2 - Mouse Genome Informatics)
(Kume et al., 2000), and the
Lim domain protein Lmx1b (Dreyer
et al., 1998).

WT1

WT1 is probably the best studied of the transcription factors expressed in
podocytes. WT1 encodes a protein with four zinc fingers that can bind
to both DNA and RNA (Call et al.,
1990; Caricasole et al.,
1996; Drummond et al.,
1994). Its loss in mice leads to complete renal and gonadal
agenesis (Kreidberg et al.,
1993), a phenotype that can be rescued in Wt1-YAC
transgenic mice, but these mice are still predisposed to developing glomerular
disease as adults (Guo et al.,
2002; Menke et al.,
2003; Moore et al.,
1999; Moore et al.,
1998; Patek et al.,
2003; Schedl and Hastie,
1998). Whether the adult-onset glomerular disease observed in
these mice reflects developmental abnormalities that are not obvious, but
nevertheless lead to pathological changes, or whether WT1 regulates the
expression of genes that are required to maintain normal glomeruli throughout
life is a matter of investigation. The latter possibility is consistent with
the emerging paradigm that glomeruli are structures that require an active
`maintenance' function throughout life.

Schematic of the slit diaphragm and other important proteins involved in
maintaining foot process assembly. Many proteins are omitted to emphasize
the two major complexes of proteins discussed in the text. For clarity, one
complex is shown within the left foot process, the other in the right foot
process. Two adjacent foot processes are shown. In the left foot process is
the nephrin-FYN-NCK complex, associated with an actin filament. In the right
foot process, the nephrin-podocin-CD2AP complex and the integrin-linked kinase
(ILK)-parvin-pinch complex associated with both α3β1 integrin and
nephrin are shown. Synaptopodin and α-actinin 4 are also shown
associated with the cytoskeleton, the latter also with integrins. Nephrin and
FAT are shown as two major proteins that bridge the space between adjacent
foot processes, although, as noted in the text, nephrin also associates with
Neph proteins. The integrin and dystroglycan complexes are shown in each foot
process. P, phosphorylation (see key for other abbreviations used in the
figure).

The targets of and functions of WT1 remain an enigma, as there are four
major splice forms of WT1 mRNA
(Haber et al., 1991), whose
encoded proteins have differing abilities to bind DNA and RNA, and different
translational start sites, indicating the possibility of many different
peptides (Bruening and Pelletier,
1996). It is therefore likely that WT1 has several functions
during early kidney and glomerular development. Numerous genes have been
suggested to be its regulatory targets, but their validity remains unclear,
either because they are not co-expressed with Wt1 in vivo, or because
mutations in these genes do not yield phenotypes that overlap with the
Wt1 mutant phenotypes in mice or humans, though it is conceivable
that, if WT1 regulates many genes, no single phenotype resulting from mutation
of a target gene would recapitulate the entire Wt1 mutant
phenotype.

With the recent strides in the ability to analyze gene expression within
the context of chromatin, the study of transcription factors has undergone
dramatic advancements that will impact the consideration of the past
literature on the function of WT1. Many studies of the molecular function of
WT1 were done using plasmids containing putative target sequences of WT1
(reviewed by Scharnhorst et al.,
2001). Even though WT1 binds to DNA containing these sequences, in
most instances they were not studied in the context of assembled chromatin in
cells that would express Wt1 in vivo. More recent studies are
beginning to use chromatin immunoprecipitation to verify WT1 target genes
(Kim et al., 2007). However,
even these studies generally use immortalized cell lines, in which WT1 might
bind to chromatin differently than it does in the developing kidney and other
tissues. WT1 also binds to RNA, associates with spliceosomes and shuttles
between the nucleus and cytoplasm, where it associates with polysomes
(Niksic et al., 2004). A
specific interaction with heterogeneous nuclear ribonuclear protein U has been
suggested (Spraggon et al.,
2007), findings that are consistent with a post-transcriptional
function for WT1 (Larsson et al.,
1995; Niksic et al.,
2004). Thus it is possible that older publications identifying WT1
target genes might have been observing post-transcriptional effects of WT1 on
putative target genes. A WT1 interacting protein (WTIP) has been shown to
shuttle between the nucleus and the membrane
(Rico et al., 2005;
Srichai et al., 2004),
suggesting WT1 may mediate signals from the extracellular environment that
regulate gene expression, adding to the enigma of WT1.

In humans, WT1 mutations that affect the zinc finger region,
particularly in the vicinity of the third zinc finger
(Barbosa et al., 1999;
Kikuchi et al., 1998;
Little and Wells, 1997;
Pelletier et al., 1991a), are
associated with two glomerulopathies, Deny-Drash Syndrome (DDS)
(Denys et al., 1967;
Drash et al., 1970), and
Frasier syndrome, which can both present early in life and cause abnormal
glomerular development. DDS is caused by mutations that eliminate or disrupt
the zinc finger regions, whereas Frasier syndrome results from an inability to
include an alternatively spliced lysine-threonine-serine (KTS) sequence after
the third zinc finger. Mice genetically engineered to exclusively express only
the +KTS or the -KTS versions of WT1 undergo normal metanephric kidney
induction, but glomeruli are mal-developed in both mutants, suggesting that
the different splice forms of WT1 may have more distinct roles in glomerular
development than in the initiation of kidney development
(Hammes et al., 2001).

WT1 mutations in humans are also associated with diffuse mesangial
sclerosis (DMS), which is characterized by an increase in ECM deposition on
the vascular side of the GBM. Since WT1 is expressed in podocytes, on
the opposite side of the GBM from the ECM deposition, this pathological
process highlights the important regulatory interactions that occur between
podocytes, on one side of the GBM, and endothelial and mesangial cells on the
other. Why excess ECM deposition is the response to presumed aberrant gene
expression in podocytes is not known, but WT1 and other transcription factors
may regulate the signals that control the ECM protein expression that
maintains a normal GBM and the matrix that surrounds mesangial cells; mutant
forms of WT1 might thus lead to either excessive or insufficient signals,
triggering mesangial cells to overexpress ECM proteins. In the most severe
instances, neonates with DDS are identified at birth, indicating that DMS may
be considered a case of abnormal development.

WT1 might also regulate the expression of factors that affect glomerular
vascular development. Indirect evidence suggests that WT1 may regulate VEGFA
in the metanephric mesenchyme during early mouse kidney development, but there
is as yet no evidence that this occurs in podocytes
(Gao et al., 2005). Transgenic
mice that express a truncated version of Wt1 develop abnormal
glomeruli with dilated capillaries (Natoli
et al., 2002).

Nephrin, a major component of the slit diaphragm complex might also be a
target of WT1, as WT1 can bind a sequence in the promoter of NPHS1
and regulate the expression of a reporter gene placed downstream of the
NPHS1 promoter (Guo et al.,
2004; Wagner et al.,
2004). Podocalyxin, a highly charged transmembrane protein that
may confer the repulsive effects that keep podocyte cell bodies separated, is
also a possible WT1 target (Palmer et al.,
2001).

POD1 and kreisler

POD1 (also known as epicardin and capsulin) encodes a basic
helix-loop-helix (bHLH) transcription factor that is expressed early in mouse
kidney development, and subsequently in the primitive podocytes of S-shaped
bodies (Quaggin et al., 1999;
Quaggin et al., 1998a).
Kreisler (MAFB) encodes a basic domain leucine zipper (bZip) transcription
factor of the MAF subfamily and is expressed in mouse podocytes of capillary
loop-stage glomeruli (Sadl et al.,
2002). It also has an important role in hindbrain segmentation
(Sadl et al., 2003).
Pod1 and kreisler mutations in mice result in similar phenotypes:
glomerular development is arrested at the single capillary loop stage
(Quaggin et al., 1999;
Sadl et al., 2002), and the
podocytes remain as columnar-shaped cells that have lost their lateral
cell-cell attachments but remain fully adhered to the GBM without any foot
processes. Thus, Pod1 and kreisler are required just prior to the
time when podocytes would normally begin migrating around the capillary loops
and assembling foot processes. Pod1 is expressed in kreisler mutant
podocytes, indicating that kreisler is likely to act either downstream or in a
separate pathway from Pod1 (Sadl
et al., 2002).

Foxc2

Foxc2 was identified during a screen for genes with enriched
expression in mouse glomeruli (Takemoto
et al., 2006). It belongs to the forkhead-domain family of
putative transcription factors and is expressed in podocytes. In
Foxc2 mutant mouse kidneys, mesangial cells cluster at the base of
the glomerular stalk, podocyte foot processes and endothelial fenestrations
are absent, and dilated capillaries are observed, similar to the other
phenotypes discussed above (Takemoto et
al., 2006).

Regulatory interactions within the glomerulus

Is the SD a signaling complex?

Nephrin is an essential component of the protein barrier and also has an
important function in signal transduction (summarized in
Fig. 5). Nephrin has multiple
tyrosine residues that are targets for phosphorylation by, for example, the
Src family kinase FYN (Verma et al.,
2006; Verma et al.,
2003). Phosphorylation at these tyrosine residues occurs
transiently during glomerular development, concomitant with the formation of
mature foot processes (Jones et al.,
2006; Verma et al.,
2006). Nephrin phosphorylation results in the recruitment of the
adaptor protein NCK and cytoskeletal reorganization in podocytes
(Jones et al., 2006;
Verma et al., 2006;
Verma et al., 2003),
supporting the hypothesis that NCK-mediated cytoskeletal organization is
related to foot process formation. The conditional mutation of Nck1
and Nck2 in podocytes in mice results in an inability to assemble
normal foot processes (Jones et al.,
2006). Interestingly, the phosphorylation of nephrin also occurs
during podocyte damage, when foot processes disassemble; this may reflect the
onset of a repair process aimed at restoring foot process structure
(Verma et al., 2006). Further
work is needed to determine whether additional adaptor proteins also bind
nephrin at other tyrosines and whether it is a substrate for other kinases, in
addition to FYN.

Other cytoplasmic proteins that associate with the SD, probably through the
cytoplasmic domain of nephrin, include podocin (encoded by the NPHS2
gene) and CD2AP, an SH3 domain-containing protein
(Boute et al., 2000;
Li et al., 2000). Podocin and
CD2AP are both required for proper SD assembly and link the SD to the
cytoskeleton (Lehtonen et al.,
2002; Roselli et al.,
2002; Schwarz et al.,
2001; Shih et al.,
2001; Yuan et al.,
2002). Mice deficient in these proteins do not develop normal foot
process structure, as in nephrin mouse mutants
(Roselli et al., 2004;
Shih et al., 1999). A
Caenorhabditis elegans homolog of podocin (mec-2)
plays a role in mechanosensation, indicating that the SD might also have a
mechanosensory function (Huang et al.,
1995). Future work should determine whether adaptor proteins, such
as NCK, also interact with SD complex proteins, such as Podocin and CD2AP, to
form a larger signaling complex.

Recently, phospholipase C epsilon (PLCE1) has been identified as
an important gene for podocyte development. Mutations in this gene were
identified in humans with end-stage kidney disease, and the knockdown of its
homolog in zebrafish leads to abnormal glomerular development within the
pronephros (Hinkes et al.,
2006). However, there is no obvious glomerular defect in mice that
carry a targeted mutation of this gene
(Tadano et al., 2005);
whether this is due to compensation from other phospholipases is not known.
PLCE1 has a cytoplasmic distribution in podocytes that begins early in nephron
development, suggesting that signaling pathways that use this enzyme may play
an important part in podocyte differentiation. Whether it is involved in
transducing signals from adhesion complexes or SD complexes remains to be
determined.

The physiology and biochemistry of ion channels that are expressed in the
tubules of the kidney have been studied extensively. By contrast, the
importance of ion channels in the glomerulus has received, until recently,
little to no attention. This has changed with the surprising discovery that a
mutation in the TRPC6 gene accounts for a portion of human familial
glomerular disease (Reiser et al.,
2005; Winn et al.,
2005). TRPC6, a member of the TRP family of cation channels, is a
calcium channel located at the SD. Although TRPC6 is not a
`developmental' gene per se, this finding hints at the possibility that ion
channels may be of significant importance in glomerular function and perhaps
in their development.

Adhesion molecules in glomerulogenesis

By immunoelectron microscopy, β1 integrin can be detected along the
basal aspect of the foot process
(Kerjaschki et al., 1989), as
expected for a GBM receptor, a finding that is inconsistent withα
3β1 integrin also being a component of the SD. Nevertheless, the
SD and the basal aspect of the foot process are in such close proximity that a
role for α3β1 integrin in regulating signaling at the SD should not
be excluded. Thus, discussing the role of adhesion molecules separately from
those of the SD may be creating an artificial distinction. Clearly the foot
process is an extremely small structure, and the distance between its lateral
components such as nephrin, podocin and CD2AP, and the molecules that
associate basally with integrin cytoplasmic domains, such as talin andα
-actinin, could probably be spanned by a small number of proteins,
depending, of course, on their particular size and shape. Moreover, as our
knowledge of the protein-protein interactions that are involved in
cytoskeletal assembly and signal transduction expands, it is becoming
increasingly apparent that the same molecules are associated with integrin
receptors for the GBM and with components of the SD complex (e.g.
integrin-linked kinase).

A comparison of normal and α3 integrin mutant mouse
glomerular development. (A,C,E) Successive stages of normal mouse
glomerular development. (A) Capillary loop stage, at which time the
podocytes (P) still resemble a columnar epithelium and are forming a
`bowl'-shaped sheet into which capillaries are beginning to branch from a
single loop into multiple loops. Scale bar: 8 μm. (B) The capillary
loop stage is relatively normal in the absence of α3β1 integrin.
(C) Intermediate stage of glomerular development, where podocytes have
begun to lose their cell-cell attachments and migrate around capillary loops.
Mesangial cells (darker nuclei) are present in the middle of the glomerulus,
where podocytes are beginning to encompass capillary loops. (D) In the
absence of α3β1 integrin, podocytes have completely lost cell-cell
attachments, and their cell bodies appear to be connected by a thin `neck' to
the basement membrane. (E) Mature normal glomerulus. (F) In the
absence of α3β1 integrin, abnormally wide capillary loops are
present, and podocytes are mainly situated in the peripherly of the
glomerulus. GC, glomerular cleft. [Reproduced from Kreidberg et al.
(Kreidberg et al., 1996).]

Integrins are heterodimeric transmembrane proteins that serve as receptors
for the ECM and that, by forming complexes with cytoplasmic proteins, form a
structural link between the ECM and the cytoskeleton. Integrins are also
involved in signaling through multiple pathways. An emerging paradigm is that
coordinated signaling that involves integrins and receptor tyrosine kinases
(RTKs) integrates information from secreted growth factors and the ECM
(Comoglio et al., 2003).α
3β1 integrin binds many ligands but is thought to function most
effectively as a receptor for certain laminin isoforms, including laminins 5,
10 and 11 (Kikkawa et al.,
1998; Kreidberg,
2000). Podocytes express some of the highest levels ofα
3β1 integrin observed among all tissues
(Korhonen et al., 1990).α
3β1 integrin is an early marker of podocyte differentiation, and
continues to be highly expressed in mature podocytes
(Korhonen et al., 1990).
Mesangial cells (discussed below) also express α3β1 integrin,
although not as highly as podocytes
(Korhonen et al., 1990).α
3β1 integrin is expressed before the podocyte shifts from laminin
1 expression to its preferred ligands, laminin 10 and 11, but whether this
shift evokes signaling through α3β1 integrin that is related to
podocyte or foot process maturation is a matter for investigation. Podocytes
cannot assemble mature foot processes in mice with a null mutation in theα
3 integrin or α5 laminin-encoding genes
(Kreidberg et al., 1996;
Miner and Li, 2000). However,α
3β1 integrin appears not to function simply as an adhesion
receptor, because, in its absence, podocytes do not detach from the GBM, but
become flattened against a fragmented GBM
(Kreidberg et al., 1996).
Perhaps α-dystroglycan (Raats et
al., 2000), another laminin receptor expressed by podocytes,
functions redundantly to α3β1 integrin by also adhering podocytes
to laminin in the GBM, such that foot processes do not detach from the GBM as
long as either integrins or dystroglycan are present. Alternatively,α
3β1 integrin might act primarily to transduce signals that mediate
the cytoskeletal organization that is involved in forming mature foot
processes. Whether these signals are induced by changes in laminin isoform
expression is not known but it remains an intriguing possibility.

α3β1 integrin is also a component of the E-cadherin-based
adherens junction. In immortalized collecting duct epithelial cells, it is
reported to stimulate cadherin-mediated cell-cell adhesion. α3β1
integrin forms a complex that includes the tetraspanin CD151, PTPμ (a
transmembrane receptor tyrosine phosphatase) and PKCβII
(Chattopadhyay et al., 2003).
This complex appears to be involved in maintaining low levels of tyrosine
phosphorylation of β-catenin. It is not clear whether α3β1
integrin fulfills this function in podocytes, and, indeed, a major role for
cadherins or β-catenin as a component of SD assembly has not been
demonstrated, suggesting that this cell-cell junction may differ significantly
from more typical cell-cell junctions, especially as it acquires its specific
function as a protein barrier. However, podocytes do lose their cell-cell
attachments more readily in the absence of α3β1 integrin
(Fig. 6), and acquire a highly
abnormal morphology that is probably incompatible with normal glomerular
development and function (Kreidberg et
al., 1996).

Integrin-linked kinase (ILK) is an important molecule that might link
integrins and associated proteins to the SD complex. ILK has serine-threonine
kinase activity (Hannigan et al.,
1996), although whether the kinase activity is required for its in
vivo functions is unknown. Recently, ILK has been shown in podocytes to
associate with parvin and pinch (Yang et
al., 2005), two adaptor proteins. This larger complex associates
with actin-binding proteins through parvin and possibly with RTKs through an
interaction between pinch and the adaptor proteins NCK1 and NCK2
(Xu et al., 2005). In
podocytes, ILK also associates with a complex that includes nephrin andα
-actinin4 (the latter belongs to the α-actinin family of
actin-binding proteins that interact with integrin-associated complexes)
(Dai et al., 2006). Point
mutations in the ACTN4 gene in humans and mice lead to glomerular
disease (Kaplan et al., 2000;
Kos et al., 2003). These
observations suggest that the ILK-pinch-parvin complex may be part of a bridge
between the SD and integrins, where it could be involved in mediating or
regulating their attachment to the cytoskeleton
(Dai et al., 2006).
Interestingly, the conditional inactivation of the ILK gene in the podocytes
of mice does not cause developmental abnormalities, but postnatally mice
develop glomerular disease beginning with loss of the foot process
architecture (El-Aouni et al.,
2006). Whether this is due to progressive gene inactivation as
mice age, or is indicative of a more important role for ILK in foot process
maintenance or repair than for their initial assembly, is not known.

Stroma in glomerular development

During kidney development, developing nephrons are surrounded by stromal or
interstitial cells. In the mature kidney, only a small number of these cells
remain, with the kidney consisting almost entirely of nephrons, with little
apparent stroma. Nevertheless, the stroma plays a crucial part in overall
kidney development (Hatini et al.,
1996). Its role in glomerular development is most notable in
chimeric mice consisting of wild-type and Pod1-/- cells,
the latter carrying a lacZ marker, which shows that
Pod1-/- cells can contribute to the glomerulus but not to
the stroma (Cui et al., 2003).
Most notably, the presence of wild-type cells in the stroma appears to rescue
the POD1 mutant glomerular phenotype, suggesting that there is a cell
non-autonomous role for POD1 in the stroma that is crucial for glomerular
development (Cui et al.,
2003).

The multiple capillary loops present in mature glomeruli appear to
originate from a single loop that invades the glomerular cleft. Whether
podocytes or mesangial cells, or possibly both, provide the crucial signals or
mechanical events that drive the establishment of the glomerular capillary
network is unclear. For example, fewer, wider-than-normal capillary loops are
present in the glomeruli of α3 integrin
(Fig. 6) or α5 laminin
mutant mice (Kreidberg et al.,
1996; Miner and Li,
2000). A recent study of mice that express mutant forms of theα
5 laminin subunit found that mesangial cells, but not podocytes, detach
from the GBM (Kikkawa et al.,
2003). [Consistent with this result, podocytes also remain
attached to the GBM in α3 integrin mutant kidneys
(Fig. 2)
(Kreidberg et al., 1996).]
This led Kikkawa et al. to hypothesize that the failure to form normal
glomerular capillary loops was due to the inability of mesangial cells to
adequately orient these loops because of their inability to securely attach to
the GBM, possibly through α3β1 integrin
(Kikkawa et al., 2003).

Signaling from podocytes

Development of the podocyte lineage is tightly linked to the
differentiation and maturation of the two other major cell compartments in the
glomerulus, the fenestrated endothelial and mesangial cells. The glomerulus is
a highly specialized capillary bed, in which podocytes function as vasculature
support cells. Podocytes produce various vascular growth factors, including
VEGFA (vascular endothelial growth factor A), VEGFC, angiopoietin 1, and
ephrin B2 (Eremina et al.,
2003; Partanen et al.,
2000; Satchell et al.,
2004; Satchell et al.,
2002; Takahashi et al.,
2001), whereas the adjacent endothelial cells express the
respective receptors for these ligands.

Podocytes begin to express all isoforms of the Vegfa gene in
S-shape bodies and continue to express them in mature glomeruli
(Kretzler et al., 1998). The
major signaling receptor for VEGFA is VEGFR2 (also known as FLK1), which is
expressed by endothelial cells as they migrate into the vascular cleft
adjacent to the podocyte precursors
(Robert et al., 1998).
Conditional gene targeting experiments in mice have shown that VEGFA
production by podocytes is essential for the formation of a functional
glomerular filtration barrier and of the fenestrated endothelial capillary
system (Eremina et al., 2003).
Loss of the VEGFA gene from developing podocytes in mice results in arrested
glomerular development and in the absence of glomerular endothelium
(Eremina et al., 2003). Less
severe reductions in Vegfa expression by podocytes, brought about by
the conditional inactivation of a single Vegfa allele, also result in
dramatic defects in the endothelial compartment that range from endotheliosis
(swelling of the endothelium) to disappearance of the endothelium followed by
rapid lysis of the mesangial cells
(Eremina et al., 2006;
Eremina et al., 2003).
Together, these results demonstrate a fine dosage sensitivity to VEGFA
production in the developing glomerulus and emphasize the role of paracrine
signaling from the podocyte to the endothelial compartment. What is less clear
is the role of juxtacrine or autocrine VEGF signaling loops within the
developing glomerulus. Although podocytes do express the VEGFR1 (FLT1),
neuropilin 1 and neuropilin 2 receptors
(Guan et al., 2006;
Villegas and Tufro, 2002), it
is not known whether they also express VEGFR2, the major receptor believed to
be responsible for VEGFA signaling. In vitro, inhibition of VEGF receptor
function affects the survival of podocytes, consistent with an autocrine
signaling loop (Foster et al.,
2003; Foster et al.,
2005). In the conditional Vegfa knockout models discussed
above, mesangial cell migration and survival is also affected. Although
mesangial cells express VEGF receptors in vitro and in diseased glomeruli,
they do not appear to express these receptors in a healthy glomerulus. Thus,
it is most likely that loss of VEGFA from podocytes affects the production of
mesangial growth factors, such as PDGFB, by the glomerular endothelium with
secondary effects on the mesangial compartment.

Multiple splice variants of the Vegfa gene give rise to a number
of pro- and anti-angiogenic isoforms. As these isoforms exhibit different
properties, they likely possess combined, as well as unique, functions. For
each major VEGFA isoform there exists a `b' isoform that arises from an
alternative distal splice site in exon 8
(Bates et al., 2002). This
results in isoforms of the same size but with a different carboxy terminus.
Investigators have shown that the 165b isoform can inhibit VEGF165-mediated
endothelial cell proliferation and migration
(Bates et al., 2002).
Intriguingly, glomerular maturation is associated with a downregulation of the
VEGF165 isoform and coincident increase in the 165b isoform
(Cui et al., 2004). It has
been suggested that failure to undergo this isoform switch may explain some of
the glomerular dysgenesis observed in individuals with Denys-Drash syndrome
caused by mutations in WT1
(Schumacher et al.,
2007).

Less is known about the signals that endothelial or mesangial cells may
exert on podocyte differentiation. Studies in zebrafish show that endothelial
cells are not required for the determination of the podocyte cell lineage, as
podocytes develop in cloche mutants that have no endothelial cells
(Majumdar and Drummond, 1999).
However, the differentiation of specialized podocyte features, such as slit
diaphragms, was not described in these mutants. Moreover, in zebrafish, in
contrast to in mammals, podocytes and tubules are derived from distinct
primordia, and caution must be used in extrapolating the results of studies of
podocyte differentiation from zebrafish to mammals, although podocytes in
zebrafish do express many of the same differentiation genes as in mammals.

Glomerular development versus damage and repair

Nephrons have a limited ability to undergo repair, confined mainly to the
proximal tubules and the ability of podocytes to re-form foot processes in the
reversible forms of glomerular disease. Podocytes have historically been
regarded as a terminally differentiated cell, and it remains unclear whether
podocytes undergo normal cell division in the mature kidney. Most studies
indicate that they have a very limited ability to proliferate, except in
certain pathological situations where podocyte proliferation replaces normal
glomerular architecture. [See Shankland
(Shankland, 2006) for a
recent complete review on podocyte injury and repair.] This is supported by
studies of experimental glomerular injury in mice with mutations in CDK
(cyclin-dependent kinase) inhibitors, such as p21 and p27, which found that
increased podocyte proliferation occurs in CDK mutant mice following renal
injury (Shankland, 2006).
However, this appears to correlate with a worsening glomerular function,
rather than with a reparative process. Thus, CDK inhibitors appear to protect
podocytes by maintaining them in a quiescent state, as a way of minimizing
irreversible damage to them in glomerular disease.

Podocyte apoptosis is also observed in experimental models of glomerular
injury (Shankland, 2006). The
possibility has been raised that the SD complex is involved in transducing
signals that affect podocyte survival. Supporting this possibility, podocyte
apoptosis is increased in mice that carry a mutation in the CD2AP gene
(Schiffer et al., 2004). In
this case, podocyte apoptosis might be a response to decreased cell-cell
adhesion, mediated through the SD.

Although podocytes do not appear to proliferate as part of a repair
mechanism, the ability to regenerate foot process architecture is an important
component of repair in glomerular disease. In some pathological situations,
foot process architecture is lost, a process referred to as effacement. In
cases where the initial damage was immune-mediated, treatment with
anti-inflammatory drugs leads to foot process restoration. In other
situations, the effacement is refractory to treatment and glomerular scarring
(glomerulosclerosis) ensues, leading to chronic renal failure. Nearly all
genetic disorders of glomerular development fall into this latter category, an
exception being the recently described mutation in the PLCE1 gene
(Hinkes et al., 2006).

One final enigma of glomerular biology is that certain mutations in mice
(or humans) that do not affect glomerular development can then lead to a loss
of foot process architecture and kidney disease in older mice, for example,
mutations in the mouse synaptopodin (Synpo) gene, which encodes an
actin binding protein. The Synpo gene knockout does not affect
initial foot process assembly, but does result in the decreased ability of
podocytes to restore foot processes in models of transient glomerular injury,
such as in the protamine sulfate/heparin model
(Asanuma et al., 2005;
Yanagida-Asanuma et al.,
2007). In this situation, foot process effacement is rapidly
induced over a matter of minutes by infusion of positively charged material
(protamine) that probably acts by masking the interactions that occur between
the highly negatively charged molecules that coat podocytes, such as
podocalyxin. Foot process architecture can be rapidly restored by the
subsequent infusion of heparin, which is negatively charged
(Seiler et al., 1975). This
demonstrates that, under experimental conditions, foot processes are dynamic
structures, and suggests that these dynamic qualities may exist during normal
in vivo function of the glomerulus, possibly as a mechanism to repair most
glomerular injury that does not otherwise come to clinical attention. What
these observations may be telling us is that the glomerulus has evolved to
withstand stress brought on by immune or environmental injury, particularly
with regard to foot process reassembly, and that there may be specific
molecules, such as synaptopodin, whose function is more important in foot
process reassembly than in their initial development. Moreover, even though it
is pleasing to think that repair mimics development, molecular mechanisms
might exist that are unique to glomerular repair. An alternate explanation is
that there is functional redundancy between many of the molecular mechanisms
that are involved in glomerular development.

Conclusion

Podocyte differentiation and damage has been the focus of much of the
research into glomerular development and disease in recent years. The emerging
paradigm that glomerular development and maintenance depends on crucial
interactions between the three major cell types of the glomerulus will serve
to re-focus future research from a podo-centric view back to one that examines
the signals that pass between these cell types, as well as between the more
distant cells within the nephron. Improved treatments of chronic and acute
kidney disease will involve regenerative therapies that produce new nephrons,
or pharmacological therapies that promote the repair of foot process
architecture and prevent glomerular scarring. Developing these treatments will
require further advancements in our understanding of the mechanisms of
glomerular development and repair.

Acknowledgments

The authors thank Wilhelm Kriz for contributing the scanning electron
micrograph and Valerie Schumacher for a critical reading of the manuscript.
This review is dedicated to the memory of Dr Paul Freeburg.